动态高压微射流技术对聚半乳糖醛酸酶活性与构象的影响
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摘要
动态高压微射流是一种新兴技术,可以对物料进行高速剪切,气穴作用等,其工作压力最高可达200 MPa。聚半乳糖醛酸酶(PG)属于果胶酶家族的一员,果胶是植物细胞壁中的一种结构性多糖,而PG主要是水解组成果胶酸的D-半乳糖醛酸α-1,4-糖苷键,从而降解植物组织。其组织软化功能在食品工业中的果汁提取和澄清中得到广泛使用。本课题通过酶活测定、圆二色谱、紫外吸收光谱和游离巯基与巯基总量的测定来研究动态高压微射流对PG酶活性和构象的影响,同时研究了热、金属离子和乙醇对PG酶的影响,揭示了PG酶活性与构象间存在着的一定的关联性。
通过动态高压微射流对PG酶进行处理,研究了PG酶活性与构象之间的关系。PG酶的最适宜反应温度为50℃。DHPM处理降低了PG酶的活性,降低后的酶活性在76.5%-94.5%。经过DHPM处理后的PG酶的构象分析显示PG酶的二级结构的β-折叠含量降低了,同时PG酶分子结构更为“松散”。其活性变化与β-折叠含量的变化呈一定程度的正相关,但不是简单的线性相关。原来包埋在分子内部的Trp和Tyr残基部分暴露出来,分子表面的二硫键被打断形成巯基,但从分子整体来看,新的二硫键形成的量要多于被打断的量。
通过热和动态高压微射流对PG酶进行处理,在50℃下保温半小时的酶活性最大,在其他温度下保温半小时的酶活性均有所降低,而在70℃下保温半小时的酶相对活性只有39.2%。在不同的温度下处理后的PG酶经过DHPM处理后的酶活性的降低程度比单独经过DHPM处理后的酶活性降低程度要大,这说明热与动态高压微射流协同处理的效果要好过单独热处理或是单独DHPM处理。PG酶的温控曲线明确表现出了PG酶的变性温度在60-65℃之间。从本实验可以看出,PG酶对温度还是敏感的,尤其是高温。
一些金属离子对PG酶起激活作用,一些起抑制作用,还有一些对酶活性没有明显影响。在本实验中,Ca2+、Ba2+、Zn2+、Mg2+对PG酶的酶活力有抑制作用,Mn2+对PG酶有激活作用。Na+对酶活力没有表现出明显的影响,不过他们的机理不尽相同。而DHPM处理对这一结果没有产生较大的影响。除了Mn2+Mn2+对经过动态高压微射流处理的PG酶没有激活作用。Ca2+、Ba2+、Na+、Zn2+ Mn2+、Mg2+均没有对PG酶的温控曲线产生较大的影响。
本实验还研究了乙醇(20%)对PG酶活性与构象的影响。PG酶在20%的乙醇溶液中活性降低了,只有未加乙醇的PG酶活性的75%。随着时间的增加,酶的活性随之降低,24 h后,酶的活性降低幅度增大,而20%的乙醇对PG酶的CD谱并未造成太大影响。因此,我们得出结论：乙醇对酶的二级结构可能未造成影响,它是通过影响酶的三级结构来影响酶的活力。乙醇对PG酶的温控曲线未造成影响,其变性温度依然处在60-65℃。
Dynamic High Pressure Microfluidization(DHPM) is a newly developed technology,which uses the combined forces of shear, cavitation, and ultra-high pressures up to 200 MPa.Polygalacturonase (PG) is a member of pectinase family that acts onα-1 4 linkages of polygalacturonic acid in pectin, a cementing substance in plant cell wall, and causes structural degradation. Its tissue-softening property is commercially exploited in the food industry in the extraction and clarification of fruit juices.This subject was to study the effect of DHPM on the activity and conformation of polygalacturonase mainly,effects of thermal、metal ions and ethanol on PG were observed at the same time by means of activity detection,far-UV circular dichroism(CD),UV absorption and sulphydryl groups detection..The result showed the relationship between activity change and conformation change of PG.
The effect of DHPM on the activity and conformation of PG was showed as follows, the optimal temperature of PG is 50℃.DHPM treatment decreased the activity of PG treated under different pressures,the residual activity of PG was in the range 76.5%-94.5%.The analysis of conformation indicated that after microfluidization treatment under different pressure,the content ofβ-sheet reduced and PG structure became looser.In addition, there were some indices that the decrease of PG relative activity was accompanied by the decrease in theβ-sheet content. Tip and Tyr residues,which buried inside the PG molecular, exposed to the solvent, S-S bonds on the surface of PG were ruptured and formed new free SH residues, while the total amount of S-S bonds new formed was more than the amount of ruptured S-S bonds.
Effect of combined thermal and DHPM on the activity and conformation of PG was studied,the result was showed as follows, The activity of enzyme incubated in 50℃for 30 min is the maxmium,the activities of PG incubated under other temperature were reducted, there was 39.2% of residual activity of enzyme incubated in 70℃for 30 min.The decrease of PG actvity under DHPM treatment after incubated under different temperature was more than the decrease of PG activity under DHPM treatment.lt demonsrated that the effect of DHPM-temperature treatment is more than DHPM or temperature treatment alone.Temperature profile of PG at the value of 218 nm showed that the denaturation temperature is in the range 60-65℃.The result indicated that PG is sensitiveness to temperature,the high temperature,specially.
Some metal ions stimulate the activity of PG,and some inhibit,others had no significance effect on the activity of PG.This subject was to study the effect of Ca2+ Ba2+、Na+、Zn2+、Mn2+、Mg2+ on the activity and conformation of PG.The result showed that, Ca2+、Ba2+、Zn2+、Mg2+ inhibited the activity of PG, Mn2+ stimulate the activity of PG,and Na+ had no effect on the activity of PG.But the mechanism of their different effect on the PG was different.DHPM treatment had no effect on the result,except Mn2+, Mn2+ had no stimulation on the activity of PG after DHPM treatment.All these metal ions had no effect on the temperature profile of PG at the value of 218 nm.
The changes of activity and conformation of polygalacturonase in ethanol(20%) were observed.The activity of PG in ethanol(20%) decreased,only 75% of redidual activity of PG leaved.The decreasement increased by the time goes.24 hours later,the decrease amplitude accelerated.However,ethanol(20%) had no conspicuous effect on the far-UV CD spectra of PG.It means that ethanol did not change the secondary structure,it had effect on the tertiary structure to change the activity of PG.Ethanol had no effect on the temperature profile of PG at the value of 218 nm,the denaturation temperature is in the range 60-65℃,which had no changed.
通过动态高压微射流对PG酶进行处理,研究了PG酶活性与构象之间的关系。PG酶的最适宜反应温度为50℃。DHPM处理降低了PG酶的活性,降低后的酶活性在76.5%-94.5%。经过DHPM处理后的PG酶的构象分析显示PG酶的二级结构的β-折叠含量降低了,同时PG酶分子结构更为“松散”。其活性变化与β-折叠含量的变化呈一定程度的正相关,但不是简单的线性相关。原来包埋在分子内部的Trp和Tyr残基部分暴露出来,分子表面的二硫键被打断形成巯基,但从分子整体来看,新的二硫键形成的量要多于被打断的量。
通过热和动态高压微射流对PG酶进行处理,在50℃下保温半小时的酶活性最大,在其他温度下保温半小时的酶活性均有所降低,而在70℃下保温半小时的酶相对活性只有39.2%。在不同的温度下处理后的PG酶经过DHPM处理后的酶活性的降低程度比单独经过DHPM处理后的酶活性降低程度要大,这说明热与动态高压微射流协同处理的效果要好过单独热处理或是单独DHPM处理。PG酶的温控曲线明确表现出了PG酶的变性温度在60-65℃之间。从本实验可以看出,PG酶对温度还是敏感的,尤其是高温。
一些金属离子对PG酶起激活作用,一些起抑制作用,还有一些对酶活性没有明显影响。在本实验中,Ca2+、Ba2+、Zn2+、Mg2+对PG酶的酶活力有抑制作用,Mn2+对PG酶有激活作用。Na+对酶活力没有表现出明显的影响,不过他们的机理不尽相同。而DHPM处理对这一结果没有产生较大的影响。除了Mn2+Mn2+对经过动态高压微射流处理的PG酶没有激活作用。Ca2+、Ba2+、Na+、Zn2+ Mn2+、Mg2+均没有对PG酶的温控曲线产生较大的影响。
本实验还研究了乙醇(20%)对PG酶活性与构象的影响。PG酶在20%的乙醇溶液中活性降低了,只有未加乙醇的PG酶活性的75%。随着时间的增加,酶的活性随之降低,24 h后,酶的活性降低幅度增大,而20%的乙醇对PG酶的CD谱并未造成太大影响。因此,我们得出结论：乙醇对酶的二级结构可能未造成影响,它是通过影响酶的三级结构来影响酶的活力。乙醇对PG酶的温控曲线未造成影响,其变性温度依然处在60-65℃。
Dynamic High Pressure Microfluidization(DHPM) is a newly developed technology,which uses the combined forces of shear, cavitation, and ultra-high pressures up to 200 MPa.Polygalacturonase (PG) is a member of pectinase family that acts onα-1 4 linkages of polygalacturonic acid in pectin, a cementing substance in plant cell wall, and causes structural degradation. Its tissue-softening property is commercially exploited in the food industry in the extraction and clarification of fruit juices.This subject was to study the effect of DHPM on the activity and conformation of polygalacturonase mainly,effects of thermal、metal ions and ethanol on PG were observed at the same time by means of activity detection,far-UV circular dichroism(CD),UV absorption and sulphydryl groups detection..The result showed the relationship between activity change and conformation change of PG.
The effect of DHPM on the activity and conformation of PG was showed as follows, the optimal temperature of PG is 50℃.DHPM treatment decreased the activity of PG treated under different pressures,the residual activity of PG was in the range 76.5%-94.5%.The analysis of conformation indicated that after microfluidization treatment under different pressure,the content ofβ-sheet reduced and PG structure became looser.In addition, there were some indices that the decrease of PG relative activity was accompanied by the decrease in theβ-sheet content. Tip and Tyr residues,which buried inside the PG molecular, exposed to the solvent, S-S bonds on the surface of PG were ruptured and formed new free SH residues, while the total amount of S-S bonds new formed was more than the amount of ruptured S-S bonds.
Effect of combined thermal and DHPM on the activity and conformation of PG was studied,the result was showed as follows, The activity of enzyme incubated in 50℃for 30 min is the maxmium,the activities of PG incubated under other temperature were reducted, there was 39.2% of residual activity of enzyme incubated in 70℃for 30 min.The decrease of PG actvity under DHPM treatment after incubated under different temperature was more than the decrease of PG activity under DHPM treatment.lt demonsrated that the effect of DHPM-temperature treatment is more than DHPM or temperature treatment alone.Temperature profile of PG at the value of 218 nm showed that the denaturation temperature is in the range 60-65℃.The result indicated that PG is sensitiveness to temperature,the high temperature,specially.
Some metal ions stimulate the activity of PG,and some inhibit,others had no significance effect on the activity of PG.This subject was to study the effect of Ca2+ Ba2+、Na+、Zn2+、Mn2+、Mg2+ on the activity and conformation of PG.The result showed that, Ca2+、Ba2+、Zn2+、Mg2+ inhibited the activity of PG, Mn2+ stimulate the activity of PG,and Na+ had no effect on the activity of PG.But the mechanism of their different effect on the PG was different.DHPM treatment had no effect on the result,except Mn2+, Mn2+ had no stimulation on the activity of PG after DHPM treatment.All these metal ions had no effect on the temperature profile of PG at the value of 218 nm.
The changes of activity and conformation of polygalacturonase in ethanol(20%) were observed.The activity of PG in ethanol(20%) decreased,only 75% of redidual activity of PG leaved.The decreasement increased by the time goes.24 hours later,the decrease amplitude accelerated.However,ethanol(20%) had no conspicuous effect on the far-UV CD spectra of PG.It means that ethanol did not change the secondary structure,it had effect on the tertiary structure to change the activity of PG.Ethanol had no effect on the temperature profile of PG at the value of 218 nm,the denaturation temperature is in the range 60-65℃,which had no changed.
引文
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